A fundamental question in biology is how the duplication, repair, and segregation of chromosomes are coordinated with each other and with progression through a cell division cycle. Faithful execution of this complex series of events is essential in order for a cell—or an organism—to pass on exact copies of its genetic material to its offspring. Homologous recombination during meiosis provides a fascinating case study to address these issues.
Meiosis is a specialized kind of cell division that is used to generate reproductive cells (sperm and eggs in mammals, or spores in fungi such as the brewer's yeast Saccharomyces cerevisiae). During meiosis, the homologous maternal and paternal copies of each chromosome are separated from one another so that the reproductive cells end up with precisely half of the starting genome complement. This way, when the reproductive cells fuse—for example, when a sperm fertilizes an egg—the normal (often diploid) genome complement is restored.
In most sexual organisms, meiotic cells carry out an intriguing process in which they damage their own DNA, and then repair the damage by copying genetic information between the maternal and paternal copies of each chromosome. This process, termed homologous recombination, is necessary to form physical connections between the homologous chromosomes that allow them to segregate accurately at the first meiotic division. If recombination fails, chromosome segregation also frequently fails, with disastrous consequences for reproductive cell formation. For example, errors in the meiotic recombination process are thought to be the underlying cause of many instances of Down syndrome, which results when a child inherits an extra copy of chromosome 21 from one of its parents.
My laboratory studies the basic mechanisms of meiotic recombination. We are interested in understanding how it works and how it is regulated so that it happens only at the right time and right place during development. We study meiosis in two organisms, mouse and S. cerevisiae, because many of the fundamental molecular mechanisms of this process are evolutionarily conserved from yeast to humans and because mouse and yeast can be experimentally manipulated in the laboratory to allow us to probe meiotic chromosome dynamics in detail.
Mechanism and Control of Meiotic Recombination Initiation
Meiotic recombination initiates with DNA double-strand breaks (DSBs) that are made by the evolutionarily conserved Spo11 protein. In budding yeast, at least nine other proteins are required along with Spo11 in order to make breaks, but the functions of these proteins are not well understood. To approach this problem, we are biochemically characterizing the DSB proteins and the multiprotein complexes they form, and using a combination of genetics and microscopy to dissect the functional relationships between these proteins and the meiotic chromosomes on which they act. In collaboration with Maria Jasin (Memorial Sloan-Kettering Cancer Center), we have also cloned and mutated the mouse homolog of the yeast SPO11 gene. This targeted mutant has given us a tool to explore the evolutionarily conserved as well as divergent aspects of Spo11 function.
We are also interested in the regulation of Spo11 activity. The potentially lethal nature of Spo11-generated DSBs puts a premium on the cell's ability to control the timing, number, and location of these DNA lesions and to control the outcome of DSB repair. These controls are understood poorly, if at all. Moreover, to form correct connections between homologous chromosomes, several processes must be spatially and temporally coordinated. For example, DNA replication and cohesion establishment must precede recombination initiation. It is not understood how cells ensure this order.
By showing that cyclin-dependent kinase (CDK) directly promotes DSB formation, we took an important step in understanding DSB regulation. We showed that CDK phosphorylates Mer2 (one of the nine yeast proteins required along with Spo11 for DSBs), identified CDK targets on Mer2, and demonstrated that mutating these sites eliminates DSB formation by disrupting interaction with other proteins. We proposed that CDK regulates Mer2 to coordinate recombination with meiotic progression. We know, however, that this is only part of the story. For example, work from other laboratories has demonstrated that the replication-regulating kinase Cdc7 is also required for DSB formation via direct modification of Mer2. We have proposed that dual regulation of Mer2 by CDK and Cdc7 provides a mechanism for controlling DSB formation relative to replication. Current work in the laboratory is testing this hypothesis.
Mapping the Distribution of Meiotic Recombination
In humans, meiotic recombination occurs preferentially within 1- to 2-kb regions (called hotspots) that are surrounded by recombinationally inert sequences. The human genome contains >15,000 hotspots, of which ~300 are utilized in any given meiotic cell. It is likely that mammalian hotspots are the sites where SPO11 preferentially forms DSBs. Understanding mammalian hotspots is important for understanding the contribution of recombination to genome evolution. The mouse is ideal for these mechanistic studies, but few hotspots have yet been identified. We are developing new methods for identifying mouse hotspots. One approach, in collaboration with the Jasin lab, uses genomic analysis to identify where crossovers occurred during derivation of inbred strains. This study resulted in the first prospective identification of novel mouse hotspots.
A second approach takes advantage of a quirk of the molecular mechanism of meiotic recombination. Spo11 breaks DNA via a topoisomerase-like reaction to generate a covalent protein-DNA intermediate with the 5' strand terminus attached to a tyrosine on the protein. Two Spo11 monomers work in concert to cleave both strands of the duplex. We recently showed in both yeast and mouse that Spo11 is removed from DSB ends by endonucleolytic cleavage, which releases Spo11 covalently attached to a short oligonucleotide. These oligos are sequence tags that mark where Spo11 made a DSB.
Starting with studies in yeast as proof of principle, we have worked out methods to sequence large numbers of these oligos. The result is an extremely high resolution map of where recombination occurs. Groundbreaking studies in a number of other labs had already developed methods for genome-wide mapping of DSBs in yeast, but because our method tells us the exact base pairs where Spo11 has cut, we are able to improve on the spatial resolution of existing maps by some two to three orders of magnitude. The increased resolution is shedding light on the distribution of hotspots in budding yeast, and we are working now to extend our mapping method to the mouse and other organisms.
Relatively little is known about the "rules" that dictate hotspot function in yeast, and even less is known in mammals. We will use the hotspots we identify to define factors that contribute to hotspot activity, e.g., sex, strain background, and local sequence differences. Through these studies, we will gain molecular understanding of this fundamental aspect of meiotic chromosome dynamics.
Controlling the Outcome of Recombination
Recombination is controlled such that each chromosome gets at least one crossover recombination product, despite a low average number of crossovers per chromosome, with multiple crossovers tending to be evenly and widely spaced. Aspects of this control have been recognized for nearly a century, since pioneering studies in the fruit fly by Hermann Muller and his colleagues. The mechanisms involved are not well understood, even today. More DSBs are formed than crossovers, so crossover control involves a decision by which a subset of DSBs becomes crossovers, while all other DSBs follow a pathway(s) that generates primarily noncrossover products. To understand the logic of this decision, we examined recombination in yeast when breaks are reduced using hypomorphic spo11 mutants. We found that crossovers tend to be maintained at the expense of noncrossovers, a previously unsuspected manifestation of crossover control that we refer to as "crossover homeostasis." Our results help to distinguish between existing models of crossover control and support the hypothesis that an obligate crossover is a genetically programmed event tied to crossover interference.
We are using crossover homeostasis and various genetic and physical assays that measure this phenomenon to explore the mechanism of crossover control. One goal is to dissect the genetic requirements of crossover homeostasis and, in particular, to test the hypothesis that homeostasis is mechanistically related to crossover interference and the obligate crossover. To do so, we are testing the effects on crossover homeostasis of a large collection of yeast mutations with known effects on other aspects of crossover control. We are also characterizing crossover control in mouse, again in collaboration with the Jasin lab.
